Background: This research aimed to analyze the psychophysiological stress response of air crews in an underwater evacuation training. Materials and Methods: We analyzed in 36 participants (39.06 ± 9.01 years) modifications in the rating of perceived exertion (RPE), subjective stress perception (SSP), heart rate (HR), blood oxygen saturation (BOS), cortical arousal (critical flicker fusion threshold, CFFT), heart rate variability (HRV), spirometry, isometric hand strength (IHS), and short-term memory (ST-M) before and after an underwater evacuation training. Results: The maneuver produced a significant (p ≤ 0.05) increase in the SSP, RPE, Mean HR and maximum HR (Max HR), and a decrease in minimum HR (Min HR) and HRV. Conclusion: An underwater evacuation training produced an increase in the sympathetic nervous system modulation, elevating the psychophysiological stress response of the air crews, not negatively affecting their cortical arousal.